Use of parasitic biological agents for disease prevention and control

ABSTRACT

The invention relates to a method of treating an excessive immune response including an aberrant/enhanced Th1 response by administering a helminthic parasite preparation in an amount sufficient to reduce the excessive immune response in an individual. This invention is generally directed to autoimmune diseases which involve an excessive immune response or an aberrant/enhanced Th1 response. More specifically, the present invention is directed to the treatment of Crohn&#39;s disease and ulcerative colitis, both known as IBD. While the present invention discloses specific information about the treatment of IBD, the disclosure is in no way limiting. Additionally, rheumatoid arthritis, type 1 diabetes mellitus, lupus erythematosis, sarcoidosis, multiple sclerosis, autoimmune thyroiditis, allergic rhinitis, colon polyps/colon cancer and asthma can be treated by the methods and compositions disclosed therein.

This application is a Divisional of Ser. No. 09/362,598, filed on Jul.28, 1999, which is a continuation in part of U.S. patent applicationSer. No. 09/209,732, filed Dec. 11, 1998, which in turn claims thebenefit of U.S. Provisional Application No. 60/070,147, filed Dec. 31,1997. The contents of each of the foregoing are incorporated herein intheir entirety.

FIELD OF THE INVENTION

This invention relates to the prevention and/or treatment of a conditionor disease state in an individual which results from an excessive immuneresponse.

BACKGROUND OF THE INVENTION

Parasites are living entities that dwell on or in other creatures duringsome part of their life cycles, drawing nourishment from the host.Parasites that inhabit the intestines have a complex interplay with themucosal immune system. They must establish a tranquil relationship withhost mucosal defenses to survive.

Helminths are elaborate multicellular worms with complex life cycles anddevelopment. The nematodes (non-segmented roundworms) and theplatyhelminths (flatworms) are the two groups of helminths that colonizethe human intestines. Perhaps more than a third of the population of theworld currently shelter one or more of these organisms. The life-timeexposure rate, however, is actually much more. The prevalence ofhelminths is highest in warm climates and in populations subject tocrowding, poor sanitation and impure food supply. Inflammatory boweldisease (IBD), rheumatoid arthritis and autoimmune diseases are rare inthese same regions.

Nematodes that frequently inhabit the human gut are Ascarislumbricoides, Enterobius vermicularis (pin worm), Trichuris trichiura(whipworm), Ancylostoma duodenale and Necator americanus (hookworms),and Strongyloides stercoralis. Trichinella spiralis infests the smallintestine briefly.

The platyhelminths include the trematodes and cestodes. The most commonadult trematodes that reside in the human intestines are Fasciolopsis,Echinostoma and Heterophyes species. Those that live in the biliarysystem include Clonorchis sinensis, Opisthorchis viverrini and felineus,and Fasciola hepatica. Schistosoma dwell in the venous system, butseveral species chronically affect the gut by the passage of eggsthrough the intestinal wall. Adult cestodes commonly infecting humansare Diphyllobothrium species (fish tapeworm), Taenia saginata (beeftapeworm), Taenia solium (pork tapeworm) and Hymenolepsis nana (dwarftapeworm).

The host acquires various helminthic species through contact with soil,food or water contaminated with the infective form of the parasite.Children most frequently harbor helminthic infections because of theirclose contact with soil and suboptimal hygienic practices. Helminthsincite an intestinal Th2 response, which can cause worm expulsion orlimit the magnitude of infection. Most children living innon-industrialized countries have these parasites. Many helminthicspecies survive for years within the gut, biliary tree or mesentericveins making thousands of eggs daily. Thus, beginning in childhood,these worms and/or their ova release molecules that bathe the intestinalmucosal surface for years inciting Th2-type inflammation. Dysregulationof the immune system leading to an excessive Th1 response may be thecause of several human diseases. Some diseases due to dominant Th1responses include IBD, rheumatoid arthritis, sarcoidosis, multiplesclerosis, insulin-dependent diabetes melitis and autoimmunethyroiditis. There is also evidence for a role of Th1-type responses inthe generation of colon polyps and colon cancer. Indeed, animal modelssuggest that excess Th1 stimulation in the intestinal mucosa can inducecolon cancer. In addition, there is evidence for Th1 involvemnet inallergic airway diseases including, but not limited to allergic rhinitisand asthma.

IBD is more common in temperate climates. It is not known what causesthe geographic differences. Observations suggest an environmentalexposure unique to temperate countries and highly industrializedsocieties predisposes to the development of IBD. An alternativeexplanation is that it is unhealthy to be raised in an “over clean”environment. It is proposed herein that the major environmental factorpredisposing to IBD is underexposure during childhood to intestinalhelminths, which promote strong Th2-type inflammation.

The frequency of CD has increased substantially over the last 40 years.It is most prevalent in temperate regions that are highlyindustrialized. This suggests that there is some critical environmentalfactor responsible for the change in frequency. Also, ulcerative colitisis rare in underdeveloped countries. It is proposed according to theinvention that the absence of exposure to intestinal helminthicinfections in childhood is an important environmental factor favoringthe development of CD and perhaps ulcerative colitis (UC).

People in industrialized countries are living in increasingly hygienicenvironments and are acquiring helminths much less frequently. Thedecreasing frequency of helminthic infections appears to correlated withthe increasing prevalence of CD. A case in point is the marked increasein the frequency of CD in young Asians and Africans after residing inIsrael for greater than 10 years. Also, the frequency of helminthicinfestation differs between the Jewish Israelis and Arabs. In 1969,stool examinations of hospitalized patients in Arab-predominant EastJerusalem contained helminthic ova over 60% of the time. The frequencyin Israeli-predominant East Jerusalem was 10% or less.

Thus, it is possible that the failure to acquire helminths and toexperience mucosal Th2 conditioning predisposes to CD and UC. There is aneed to combat CD and UC by re-colonization of the gastrointestinaltract with these organisms which may afford protection.

One object of the invention is to prevent or treat an excessive immuneresponse in an individual. The excessive or aberrant immune response canbe caused by an autoimmune disease, for example, IBD, rheumatoidarthritis, type 1 diabetes melitis, lupus erythematosis, sarcoidosis andmultiple sclerosis. It is also an object to prevent or treat theexcessive or aberrant immune response of, for example, asthma, allergicrhinitis, and colon polyps/colon cancer.

Further, it is an object of the instant invention to provide a method ofvaccinating an individual against autoimmune disease, for example, IBD,rheumatoid arthritis, type 1 diabetes melitis, lupus erythematosis,sarcoidosis, multiple sclerosis, and autoimmune thyroiditis. It is alsoan object of the invention to provide a method of vaccinating anindividual against allergic rhinitis, asthma and colon polyps/coloncancer.

It is an object of the present invention to provide a method of creatingan immune environment in an individual that is conducive to amelioratingIBD.

It is an object of the present invention to provide a pharmaceuticalcomposition comprising a helminthic parasite and a pharmaceuticallyacceptable carrier.

It is a further object of the present invention to provide apathogen-free helminthic parasite and an acceptable pharmaceuticalcarrier.

It is another object of the present invention to provide a method ofpreparing pathogen-free helminthic parasites.

SUMMARY OF THE INVENTION

The instant invention presents a method of preventing or treating anexcessive immune response in an individual by administering an effectiveamount of an helminthic parasite preparation to reduce the excessiveimmune response in the individual.

The invention encompasses a method of treating an excessive immuneresponse, including an aberrant/enhanced Th1 response, comprisingadministering a helminthic parasite preparation in an amount sufficientto reduce the excessive immune response.

The invention also encompasses a method of treating a disease involvingincreased Th1-type cytokine levels, such method comprising administeringa helminthic parasite preparation in an amount sufficient to reduce thelevel of Th1 type cytokines.

As used herein, the term “helminth parasite preparation” refers to anyone of a whole parasite, a parasite extract, parasite eggs, parasite eggextract, parasite larvae, parasite larvae extract, parasite cercariaeand parasite cercariae extract.

The helminthic preparation may be selected from the group consisting ofhelminiths that naturally colonize humans and helminths that colonizeanimals but may protect humans from an excessive Th1 response.

In other preferred embodiments of the invention, the helminthic parasiteis a nematode, and may be selected from the group such as Ascarislumbricoides, Enterobius vermicularis, Trichuris trichiura, Ancylostomaduodenale and Necator americanus, Strongyloides stercoralis andTrichinella spiralis.

In other preferred embodiments of the invention, the helminthic parasiteis a platyhelminth, and may be selected from the group consisting oftrematodes and cestodes, such as Fasciolopsis, Echinostoma andHeterophyes species, Clonorchis sinensis, Opisthorchis viverrini,Opisthorchis felineus, Fasciola hepatica, Schistosoma species,Diphyllobothrium species, Taenia saginata, Taenia solium andHymenolepsis nana.

In other preferred embodiments, the helminthic parasite is selected fromthe group consisting of filarial parasites and lung flukes.

In additional embodiments, the helminthic parasites are selected fromthe group consisting of Trichuris muris, Trichinella spiralis,Nippostrongylus prasiliensis, Heligmonsomoides polygyrus, Hymenolepsisnanan, Angiostrongylus species, Trichuris suis, Ascaris suum, Trichurisvulpis, Toxocara species, Gnathostoma species, Ancylostoma species,Anisakis species and Pseudoterranova species.

The invention also encompasses a method of preventing or treating anautoimmune disease in an individual comprising administering ahelminthic parasite in an amount sufficient to prevent or treat theautoimmune disease in an individual.

As used herein, the term “autoimmune disease” refers to diseases such asIBD, rheumatoid arthritis, type 1 diabetes mellitus, lupuserythematosus, sarcoidosis, multiple sclerosis, autoimmune thyroiditis,allergic rhinitis and asthma.

The “effective dosage” amount of the helminthic parasite preparation isdependent upon the specific cause for the excessive or aberrant immuneresponse, and is addressed in detail below.

As used herein, the term “excessive” or “aberrant” immune responserefers to a Th1 response in which the activity of T helper 1 cells iselevated in an individual relative to the activity of such cells in anindividual who is not affected by the disease. Typically, the elevationof the Th1 response in the diseased individual will be at least 2-fold,and possibly 5-fold-10-fold above the Th1 response in an individual whois not diseased. Th1-type inflammations produce large amounts of IFN-γand TNFα, which in turn stimulate a strong cellular immune reaction.These are some of the cytokines that may be measured to indicate anexcessive or aberrant Th1 response, as described in detail hereinbelow.

Preferably, the helminthic parasite is selected from the groupconsisting of parasite, parasite extract, parasite eggs, parasite eggextract, parasite larvae, parasite larvae extract, cercariae andcercariae extract.

The invention also encompasses a method of treating IBD comprisingadministering a helminthic parasite preparation in an amount sufficientto reduce IBD.

In a preferred embodiment, the invention also encompasses a method oftreating IBD comprising administering a helminthic parasite preparationin an amount sufficient to reduce IBD wherein the IBD is Crohn's diseaseor ulcerative colitis.

The invention also encompasses a method of treating multiple sclerosiscomprising administering a helminthic parasite preparation in an amountsufficient to reduce multiple sclerosis.

The invention also encompasses a method of vaccinating an individualagainst a disease involving an excessive immune response comprisingadministering a helminthic parasite preparation in an amount sufficientto prevent the excessive immune response.

The invention also encompasses a pharmaceutical composition comprising ahelminthic parasite preparation, and a pharmaceutically acceptablecarrier.

The invention also encompasses a method of producing a helminthicparasite preparation comprising isolating a helminthic parasite from thestool of a mammal housed in a specific pathogen-free environment, andformulating a pharmaceutical composition comprising said parasite and apharmaceutically acceptable carrier.

The invention also encompasses a method of producing a helminthicparasite preparation from animal or plant tissue, or soil in apathogen-free environment, and formulating a pharmaceutical compositioncomprising said parasite and a pharmaceutically acceptable carrier.

The invention also encompasses a method of treating a disease involvingincreased infiltration of inflammatory cells into the CNS by decreasingthe number of inflammatory cells infiltrating the CNS, the methodcomprising administering to a mammal a helminthic parasite preparationin an amount sufficient to decrease the number of inflammatory cells inthe CNS.

The invention also encompasses a method of treating an autoimmunedisease by inducing Th2 cytokines, the method comprising administeringto a mammal a helminthic parasite preparation in an amount sufficient toinduce one or more Th2 cytokines.

As used herein, the term “Th2 cytokine” or “Th2-type cytokine” refers toa cytokine produced in a Th2 response. Th2 cytokines include, forexample, IL-4 and IL-5.

As used herein, the term “Th1 cytokine” or “Th1-type cytokine” refers toa cytokine produced in a Th1 response. Th1 cytokines include, forexample, IFN-γ and TNF-α.

As used herein, the term “increased Th1 cytokines” or “increasedTh1-type cytokines” refers to a situation in which the level of a Th1cytokine is increased relative to baseline levels. The level of a Th1cytokine may be said to be increased if there is at least a 2-foldincrease over the baseline level, and possibly as much as a 5- to10-fold increase or more.

As used herein the term “inducing Th2 cytokines” refers to a situationin which the level of a Th2 cytokine is caused to increase relative to abaseline level. The level of a Th2 cytokine may be said to be “induced”if there is at least a 1-fold increase over the baseline level, andpreferably at least a 2-fold to 8-fold increase or more.

As used herein, the term “disease involving increased infiltration ofinflammatory cells” refers to IBD, colon polyps/colon cancer, rheumatoidarthritis, type 1 diabetes mellitus, lupus erythematosus, sarcoidosis,multiple sclerosis, autoimmune thyroiditis, allergic rhinitis andasthma.

As used herein, the term “increased” in reference to infiltration ofinflammatory cells or cytokines refers to a situation in which thenumber of inflammatory cells or the level of cytokines at a particularsite is elevated above baseline levels. The elevation or increase may beby 5%, 10%, 20%, 50%, 100% or more.

As used herein, the term “baseline level” refers to the level of acytokine or cell type associated with tissues which are not affected bya disease characterized by an excessive immune response.

As used herein, the term “decrease” in reference to the number ofinflammatory cells or the level of cytokines refers to a lessening inthe number of inflammatory cells or in the amount of cytokines at a sitecharacterized by infiltration of inflammatory cells or increasedcytokines. The lessening or decrease may be by 5%, 10%, 20%, 50% ormore, up to and including as much as 100%.

As used herein, the term “inflammatory cells” refers to cells involvedin inflammation. Examples of cells involved in inflammation aregranulocytes (such as neutrophils or polymorphonuclear leukocytes,eosinophils and basophils) as well as mast cells, lymphocytes, NK(natural killer) cells and macrophages/monocytes.

The invention also encompasses a method of screening a helminthicparasite preparation for one or more components that reduce an excessiveTh1 immune response, the method comprising the step of assaying afraction of a helminthic parasite preparation to detect a biologicalactivity that reduces an excessive Th1 immune response.

In a preferred embodiment, the method further comprises, prior to theassaying step, the steps of: i) providing a helminthic parasitepreparation; and ii) fractionating the helminthic parasite preparation.

In a preferred embodiment, the helminthic parasite preparation isprepared from one of the group consisting of helminthic parasite eggs,larvae, cercariae and adult worms.

In another preferred embodiment, a fraction containing one or morecomponents that reduce an excessive Th1 immune response is subjected tothe further step of fractionating and assaying to identify asub-fraction containing one or more components that reduce an excessiveTh1 immune response. It is also preferred that the further fractionatingand assaying steps are repeated at least once.

It is further preferred that the fractionation is performed using one ormore chromatographic separation techniques.

In a further preferred embodiment, the one or more chromatographicseparation techniques is/are selected from the group consisting of:column chromatography; HPLC, FPLC, matrix-affinity chromatography;reverse-phase chromatography; and electrophoretic separation.

In one embodiment, the assaying comprises an in vitro assay.

In another embodiment, the assaying comprises an in vivo assay.

As used herein, the term “a component that reduces an excessive Th1immune response” refers to a portion of a helminthic parasitepreparation (including, but not limited to, one or more peptides,proteins, carbohydrates, nucleic acids, lipids, hormones, fatty acids,polyamines, prostaglandins, leukotrienes, glycoproteins and glycolipids)made from helminthic parasite eggs, larvae, cercariae or adult wormsthat has activity in in vitro or in vivo assays for either a reduced Th1responses or an induced Th2 response.

As used herein, the term “helminthic parasite preparation” refers to apreparation of helminthic parasite (eggs, larvae, cercariae or adultworms) that has been subjected to homogenization using mechanical,ultrasonic, thermal (heating or cooling or both) or other disruptivemeans, and/or that has been subjected to purification.

As used herein, the term “biological activity that reduces an excessiveTh1 immune response” refers to an activity contained within a helminthicparasite preparation that may be detected by its ability to reduce a Th1immune response or an indicator of a Th1 immune response in an assaymeasuring Th1 immune response or indicators of a Th1 immune response.

As used herein, the term “fractionating” refers to the process ofdividing a helminthic homogenate or fraction of a homogenate intosmaller sub-portions or fractions on the basis of some physical,chemical or biochemical property.

As used herein, “chromatographic separation technique” refers to atechnique that separates components of a mixture based on size, charge,molecular weight, hydrophilic/hydrophobic interactions, solventinteractions and/or specific binding interactions.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The figure describes the concentrations of IFNγ, IL-4 and IL-5measured in spleen cell supernatants of mice infected with M. avium, S.mansoni or both organisms. Splenocytes (4×10⁵/well) were cultured invitro for 48 h at 37° C. in 200 μl of medium in the presence or absenceof optimal concentrations of PPD or SEA. Cytokine secretion wasquantified by ELISA.

FIG. 2. The figure describes the concentrations of IFNγ and IL-4 ingranuloma cell supernatants of mice infected with M. avium, S. mansonior both organisms. Granuloma cells (4×10⁵/well) in 200 μl of medium werecultured in vitro at 37° C. for 48 h in the presence or absence of theoptimal concentration of PPD or SEA. Cytokines were quantified by ELISA.

FIG. 3. The figure describes the serum IgG1, IgE and IgG2a levelsmeasured in mice infected with M. avium, S. mansoni or both(concurrent). Immunoglobulin concentration was determined by ELISA.

FIG. 4. The figure shows that IL-4 treatment can cure mice of a chronicH. polygyrus infection. Mice received three injections of an IL-4complex starting 12 days before killing. Data are means+/−SE of multipledeterminations.

FIG. 5. The figure shows the effect of schistosome ova injection(Protocol #1) on induction of EAE. Schistosome ova-injected (solid line)or control SJL/J (dotted line) mice were immunized subcutaneously withPLP139-151 in CFA. Mice were examined daily and scored for EAE. Data arepresented as means+/−SEM.

FIG. 6. The figure shows the effect of increased Schistosome ovainjection (Protocol #2) on induction of EAE. Schistosome ova-injected(solid line) or control SJL/J (dotted line) mice were immunizedsubcutaneously with PLP139-151 in CFA. Mice were examined daily andscored for EAE. Data presented are means+/−SEM.

FIG. 7. The figure shows the effect of Schistosome ova injection on theprogression of EAE. Schistosome ova-injected (solid line) or controlSJL/J (dotted line) mice were immunized subcutaneously with PLP139-151in CFA. Mice were examined daily and scored for EAE. Data presented aremeans+/−SEM.

FIG. 8. The figure shows histopathological analyses of spinal cord fromnormal (A) or schistosome-injected (B) mice with EAE. Mice were killed14 days following immunization. Spinal cords were harvested, fixed in10% formalin and embedded in paraffin. Tissue sections 8 micrometersthick were stained with hematoxylin and eosin (HE).

FIG. 9. The figure shows the results of ELISA assays of IL-4 (A), IL-2(B) and IFN-γ in the supernatants of cultures of activated splenocytes.Open bars indicate values obtained with cells from control(non-Schistosome-immunized, EAE) mice and the filled bars indicatevalues obtained with cells from Schistosome-immunized EAE mice.

FIG. 10. The figure shows the results of an ELISPOT assay in anallospecific Balb/c versus C57BL/6 recall response after 7 days with(bottom panel) or without (upper panel) Th2 manipulation (α-IL-4,α-IFNγ).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the recognition that diseases thatinvolve excessive or aberrant Th1 immune responses are treatable byadministration of a helminthic parasite preparation.

The invention relates to a method of treating an excessive immuneresponse including an aberrant/enhanced Th1 response by administering ahelminthic parasite preparation in an amount sufficient to reduce theexcessive immune response in an individual. This invention is generallydirected to autoimmune diseases which involve an excessive immuneresponse or an aberrant/enhanced Th1 response. The present invention isdirected to the treatment of diseases such as Crohn's disease andulcerative colitis, both known as IBD, multiple sclerosis (MS),Rheumatoid arthritis, type 1 diabetes mellitus, lupus erythematosis,sarcoidosis, autoimmune thyroiditis, allergic rhinitis and asthma.

The entire disclosure of each of the literature references citedhereinafter are incorporated by reference herein.

Helminthic Parasites Useful According to the Invention

In defining a helminthic parasite, there are two groups. The first groupis helminthic parasites that naturally colonize humans and the secondgroup is helminthic parasites that colonize animals, but may affordprotection to humans.

In the first group, helminthic parasites are elaborate multicellularworms with complex life cycles and development. The nematodes(non-segmented round worms), and the platyhelminths (flat worms) are twogroups of helminths that colonize the human intestines. In accordancewith the present invention, any one of a number of helminth parasitesthat naturally colonize humans or animals will provide the intendedresults.

Nematodes that frequently inhabit the human gut are Ascarislumbricoides, Enterobius vermicularis (pin worm), Trichuris trichiura(whipworm), Ancylostoma duodenale and Necator americanus (hookworms),and Strongyloides stercoralis. Trichinella spiralis infests the smallintestine briefly.

The platyhelminths include the trematodes and cestodes. The most commonadult trematodes that reside in the human intestines are Fasciolopsis,Echinostoma and Heterophyes species. Those that live in the biliarysystem include Clonorchis sinensis, Opisthorchis viverrini and felineus,and Fasciola hepatica. Schistosoma dwell in the venous system, butseveral species chronically affect the gut by the passage of eggsthrough the intestinal wall. Adult cestodes commonly infecting humansare Diphyllobothrium species (fish tapeworm), Taenia saginata (beeftapeworm), Taenia solium (pork tapeworm) and Hymenolepsis nana (dwarftapeworm).

Other helminths of interest include the filarial parasites and the lungflukes. These do not have a gut phase, but stimulate strong Th2-typeresponses.

The second general group of helminthic parasites that can be utilized inthe present invention include helminths that colonize animals, but mayafford humans protection against Th1-mediated diseases. These includeTrichuris muris (mouse whipworm), Trichinella spiralis, Nippostrongylusbrasiliensis, Heligmonsomoides polygyrus and Hymenolepsis nana, all ofwhich are intestinal helminths that infect mice. Additionally,Angiostrongylus is a rat helminth. Trichuris suis and Ascaris suum arepig helminths that can infect humans. Trichuris vulpis, Toxocaraspecies, Gnathostoma, and Ancylostoma are dog or cat helminths that alsocan infect humans. Anisakis and Pseudoterranova are nematodes of marinemammals that can transmit to humans. Bird schistosomes can transientlyinfect humans. Such schistosomes include S. douthitti, Trichobilharziaocellata, T. stagnicolae, T. physellae, and Gigantobilharzia huronensis.

Diseases Treatable According to the Invention A. Inflammatory BowelDiseases (CD and UC)

Epidemiological data suggest a genetic susceptibility to the developmentof Crohn's disease (CD) and ulcerative colitis (UC). The incidence of CDin industrialized societies has increased from the 1950s until the mid1980s, and now is from 1 to 8 per 100,000 persons per year. Thissuggests that unknown changes in our environment have effected thefrequency of CD.

While the cause of IBD remains undetermined, it is presumed to resultfrom dysregulation of the intestinal mucosal immune system. Inflammatorycells in the mucosa normally protect us from luminal contents. Thishighly effective chronic inflammation is tightly controlled to limittissue injury. IBD may result from inappropriately vigorous immuneresponses to luminal factors. CD appears to be an overly vigorousTh1-type inflammation that produces IFN-γ and TNFα. The nature of UC isless well defined.

There are several animal models of chronic intestinal inflammation, forexample, as reported by Elliott et al. (Elliott et al., 1998,Inflammatory Bowel Disease and Celiac Disease. In: The AutoimmuneDiseases, Third ed., N. R. Rose and I. R. MacKay, eds. Academic Press,San Diego, Calif.). An important advance is the recent discovery thatsome mice with genetically engineered gene deletions can develop chronicbowel inflammation similar to IBD, see Elson et al., 1995,Gastroenterology 109:1344. These include mutant mice bearing targeteddeletions for IL-2, IL-10, MHC class II or TCR genes among others. Usingsome of the models, Berg et al., 1996, J. of Clin. Investigation98:1010, Ludviksson et al., 1997, J. of Immunol. 158:104, and Mombaertset al., 1993, Cell 75:274 have shown that a dysregulated immune systemitself can mediate intestinal injury. The mucosal inflammation ofseveral of these models generates large amounts of IFN-γ and TNF-αsuggesting that excess production of Th1-type cytokines is one commonmechanism underlying the pathogenesis of disease. Also, blocking Th1circuitry prevents the inflammation. CD is a Th1 response. Thus, thesemodels may have direct implications regarding the immunopathology ofthis human disease process.

B. Rheumatoid Arthritis (RA)

RA is a chronic disease featuring persistent inflammatory synovitis,usually involving peripheral joints in a symmetric distribution. Thisinflammation can lead to bone erosions, cartilage damage and jointdestruction. It is an affliction of about 1% of the population. Theprevalence increases with age, and women are affected more frequentlythan men. The propagation of RA is an immunologically mediated eventdriven by CD4+ Th1 cells.

C. Insulin-Dependent, Juvenile Diabetes Mellitus (DM)(type 1)

Type 1 DM is a disease that usually begins during early adulthood andthat results from the inability to produce insulin in response toincreasing blood sugar. This persistent high blood sugar and inabilityto properly metabolize glucose causes metabolic disturbances thateventually damage the eyes, kidneys, heart and other organs. Takinginsulin parenterally can partially control these metabolic problems.Type 1 DM results from an autoimmune attack on the pancreatic betacells, which are the source of insulin. Activated macrophages andcytotoxic T cells surround and destroy the pancreatic beta cells.Genetic susceptibility and poorly defined environmental events triggerthe disease process.

D. Lupus Erythematosus (LE)

LE is a systemic autoimmune disease that is most frequent in women ofearly to middle adulthood. The tissue damage is caused by autoantibodiesand hyper-reactive regulatory T cells. The abnormal immune responseallows sustained production of pathogenic autoantibodies and immunecomplexes. This leads to damage of the musculoskeletal, cutaneous,hematologic, renal and other systems. The abnormal immune responseprobably depends upon the interaction of multiple hereditary andenvironmental factors.

E. Sarcoidosis

Sarcoidosis is a chronic granulomatous disease of the lungs and otherorgans of unknown cause. Most patients present between the ages of20-40. The most frequent symptom is shortness of breath. The diseaseresults from an exaggerated Th1-type, cellular immune response, probablyto a limited number of antigens. Sarcoidosis develops throughout theworld, and afflicts all races. However, there is remarkable diversity ofthe prevalence of sarcoidosis among certain ethnic and racial groups.For instance, the disease is rare in Poland, Southeast Asia and India.

F. Multiple Sclerosis (MS)

MS is a chronic relapsing, multifocal inflammatory disorder of thecentral nervous system that leads to focal demyelination and scarring ofthe brain. It is a frequent disease affecting about 350,000 Americansand which begins during early to middle adulthood. MS is an autoimmunedisease mediated at least in part by Th1 cells. The lesions of MSresemble those induced by delayed hypersensitivity responses thatcontain activated T cells and macrophages. It is a disease of temperateclimates, increasing in prevalence with distance from the equator.

G. Autoimmune Thyroiditis

As implied by its name, autoimmune thyroiditis is an autoimmune diseaseresulting in inflammation of the thyroid gland. The disease results fromTh1-type inflammation, and is marked by the presence ofantithyroglobulin autoantibodies. The disease may be studied in a ratmodel as described, for example, by Seddon & Mason, 1999, J. Exp. Med.189: 279.

H. Colon Polyps/Colon Cancer

Adenomatous polyps of the colon and rectum are common benign neoplasmsthat are usually asymptomatic. They may be single or multiple and occurmost frequently in the sigmoid and rectum. In humans, diagnosis isestablished by sigmoidoscopy, double-contrast barium enema, andcolonoscopy. Larger adenomatous polyps pose a high risk of malignanttransformation. Villous adenomas are usually sessile and becomemalignant in 10% of cases if they are 2 cm in diameter or less, and inup to 50% of cases if they are larger.

The incidence of polyps increases with age in humans. Colon polyps andcancer are exceedingly rare in underdeveloped countries. Animal modelshave provided evidence that excess Th1-type stimulation in theintestinal mucosa can induce colon cancer (see, for example, Rudolph etal., 1995, Nature Genet. 10: 143; Van Kruiningen et al., 1983, J.Compar. Pathol. 93: 275; Van Kruiningen, 1972, Gastroenterology 62:1128).

I. Allergic Airway Diseases

There is evidence for a role of Th1-type triggers in allergic airwaydiseases such as allergic rhinitis (hay fever) and asthma. Both of thesediseases are rare in underdeveloped countries, while 20% of the U.S.population suffers from hay fever, and asthma cases have increaseddramatically in recent decades.

Helminthic Parasite Preparations According to the Invention A. ViableOrganism Vaccines

Viable helminthic parasite organisms may provide the most profound Th2mucosal conditioning because of their relative longevity as compared tocomponent vaccines. Viable organisms can be administered in either egg,larval, cercarial, or encysted larval forms depending on the helminth.Helminths that can colonize humans and a preparatory animal may beutilized.

The preparatory animal may need manipulation to allow high patency bythe helminthic. Such manipulation can include treatment with agents thatare immunosuppressive like glucocorticoids or azathioprine; agents thatimpede Th2 effects like anti-histamines, anti-cytokines, or recombinantcytokines; and agents that influence intestinal motility likeanti-cholinergics or opiates. The animal's diet will be altered toreduce coarse fiber content. The animal may be a rat, pig, hamster, birdor other preparatory animal.

Preparatory animals are raised in specific pathogen-free (SPF)environments according to methods known in the art. They are tested toensure absence of human bacterial, mycobacterial, and viral pathogens.

Eggs: Some intestinal helminths are acquired by ingestion of viableeggs. Helminths are maintained in SPF preparatory animals, for example,SPF pigs. To harvest eggs, the animals are given a special diet low incoarse fiber. Animals are given an oral purgative to induce defecation.Stool is collected and enzymatically digested to free eggs. Eggs areisolated from liquefied stool by sedimentation, floatation on densitygradients, screen filtration, Visser filtration, or centrifugalelutriation. Preservation of eggs varies with the helminth used. Eggsfrom helminths that are resistant to dessication are dried, compoundedwith inert material, incorporated into an enteric capsule, andrefrigerated. Eggs from helminths that are susceptible to dessicationare preserved by refrigeration in liquid medium or by addingcryoprotectant and freezing in liquid nitrogen. Viable eggs are washed,mixed with chilled lactose-free pudding or other vehicle at the locationfor delivery. Eggs stored in glycerol-based cryoprotectant may notrequire washing. Eggs from each lot are tested for hatching rate todetermine effective dosing. Eggs from each lot are tested for absence ofbacterial and viral pathogens.

Larvae: Some helminths (i.e. hookworms) require a soil maturation phasebefore they can colonize humans. Eggs from these agents will beincubated under optimal conditions to mature the embryo, or hatch theegg and provide larval forms. Patients can be inoculated by subcutaneousinjection of viable larvae.

Cercariae: Some helminths have complex life cycles that utilize anintermediate host. The intermediate host sheds the form able to colonizehumans. Cercariae are the form for trematode helminths (i.e. flukes)shed by intermediate hosts like snails. Cercariae are isolated fromcolonized snails grown in SPF conditions. Cercariae are washed. Thesemay be preserved by adding cryoprotectant and freezing in liquidnitrogen. Thawed or fresh cercariae are washed and injectedsubcutaneously to inoculate patients. Samples from each lot are testedfor absence of pathogens and to determine effective dose.

Encysted Larvae: Some helminths (e.g. tapeworms) form encysted larvae orcysticerci in intermediate hosts. It is the encysted larval form thatinitiates human colonization. Encysted larva are removed fromintermediate hosts, for example, cattle or fish or plants grown in SPFconditions. Cysts are washed free of remaining host tissue. Cysts may bepreserved by adding cryoprotectant and freezing in liquid nitrogen.Cysts are thawed or used fresh, washed, mixed with chilled lactose-freepudding or other vehicle at the location for delivery and fed toindividuals. Samples from each lot are tested for absence of pathogensand to determine effective dose.

B. Non-Viable Component Vaccines

Non-viable components of helminthic parasites provide sufficient Th2conditioning of the immune response to prevent Th1-mediated pathology.Non-viable components are derived from eggs, larvae or adult worms.

Non-viable, intact schistosome eggs produce a strong Th2 response. Eggsare isolated from livers of preparatory animals (i.e. hamsters) grownunder SPF conditions. Eggs are isolated by a method modified from thatoriginally described by Coker and Lichtenberg, 1956, Proceedings of theSoc. For Exp. Biol. & Med. 92:780. The modifications consist of usingphosphate buffered saline with glucose instead of 1.7% saline forincubation and washing steps along with decreasing the autolyticdigestion time. These changes promote isolation of viable eggs. Themethod is as follows.

Infect golden hamsters with 1000 to 1500 cercariae. Allow the infectionto mature (6 to 7 weeks). Remove the livers from the animals and placein 600 mOsm sterile phosphate buffered saline containing 5% glucose, 100U/ml penicillin and 100 mg/ml streptomycin. The livers are allowed toautodigest for 24 hours at room temperature. Pulse homogenize the liversat low speed for 3 minutes in a cold Waring blender. Incubate thehomogenate with collagenase (2 mg/ml) and trypsin (2 mg/ml) at 32° C.for one hour. Filter the homogenate through 50 and 80-100 mesh sieves toremove clumps of tissue and debris. Recover the eggs from the filtrateby passing over a 325 mesh sieve. The eggs will not pass through thescreen. Flush the eggs off of the screen and into a 50 ml polypropylenecentrifuge tube. Wash the eggs by repeated low speed (400×g)centrifugation in sterile phosphate buffered saline with 5% glucose. Theeggs must be free of any collagenous debris. Count an aliquot of eggs ina 1 ml Sedwick chamber to determine total egg number.

Isolated eggs are suspended in saline and flash frozen in liquidnitrogen without cryoprotectant. This kills the egg. Thawed eggs areinjected subcutaneously, intramuscularly or intravenously, or at sitesof Th1 inflammation to elicit strong Th2 responses. Eggs from otherhelminths may also be utilized.

Component vaccines may also be used that employ proteins, lipids, orcarbohydrates isolated from parasite eggs. An example is schistosomesoluble egg antigens (SEA). The method for preparing schistosome eggantigen has been previously described by Boros and Warren, 1970, J. ofExperimental Med. 132:488 and is briefly given below.

Washed eggs are resuspended at 50,000 eggs/ml of phosphate bufferedsaline. This is transferred to a glass tissue homogenizer. The eggs arehomogenized on ice. To insure that all shells are broken and miracidiaare disrupted, an aliquot (5 ml) is removed for microscopic inspection.Transfer the homogenate to ultracentrifuge tubes. Centrifuge at100,000×g for 2 hours at 4° C. Recover the aqueous fraction (SEA) anddetermine the protein content. Store the SEA in small aliquots at −70°C. This method may require modification to isolate the parasite eggproducts that most strongly promote Th2 conditioning, i.e., to achievean optimal effective concentration, (100 μg SEA or 10,000 ova/animal).Eggs or soluble egg components are used to initiate Th2 responses or toboost Th2 responses previously initiated by colonization with viablehelminths. Eggs or egg components are tested to confirm the absence ofpathogens and endotoxin.

Component vaccines can also be developed from larvae and adult worms ofhelminthic parasites. Larvae or worms are isolated from preparatoryanimals grown in SPF conditions. Vaccines that employ non-viable intactorganisms or proteins, lipids, or carbohydrates isolated from thehelminth are prepared and utilized in a manner similar to thatpreviously described for helminth eggs.

C. Maintenance of Helminth Organisms

Helminths are cycled through intermediate and preparatory animals grownin SPF conditions. Samples of helminth populations are tested to ensurephenotypic stability such as colonization rates, fecundity, andsusceptibility to anti-helminthics.

Dosage, Administration and Pharmaceutical Formulations

Depending upon the helminthic parasites selected, treatment is deliveredby one of several approaches. Individuals in need of treatment receivethe infective form of the parasite (egg, cercariae or larvae) orally orparenterally depending upon the natural life cycle of the parasiteselected. Alternatively, soluble worm or egg extracts can be givenorally or parenterally to induce Th2 responses.

With regard to intestinal and liver helminths and schistosomes, theybegin producing ova that appear in the stool about 30-60 days afterinoculation. Quantifying the eggs in the stool proves satisfactory forassessing adequacy and intensity of infection. Aliquots of stool areprocessed by sucrose floatation to determine the total number of eggs ineach specimen. Flotation over sucrose solution is a method frequentlyused to isolate eggs from stool for accurate counting as reported byKoontz and Weinstock, 1996, Gastroenterology Clinics of N. America,25:435.

The helminthic parasite compounds of the invention may be formulated foradministration in any convenient way, and the invention thereforeincludes within its scope pharmaceutical compositions comprising thehelminthic parasite compound in accordance with the invention adaptedfor use in humans. Such compositions may be presented for use inconventional manner with the aid of any necessary pharmaceuticalcarriers or excipients.

The helminthic parasite compound according to the invention may beformulated for injection, and therefore, vaccine use, and may bepresented in unit dose form in ampules, or multidose containers, ifnecessary, with an added preservative. The compositions may also takesuch forms as suspensions, solutions or emulsions of oily or aqueousvehicles and may contain formulatory agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the helminthicparasite may be in powder form or reconstituted with a suitable vehicle,e.g. sterile-pyrogen-free water, before use.

If desired, such powder formulation may contain an appropriate non-toxicbase in order to ensure that the powder is reconstituted with water, thepH of the resulting aqueous formulation being physiologicallyacceptable.

The helminthic parasite compounds may also be formulated assuppositories, e.g. containing conventional suppository bases such ascocoa butter or other glycerides.

The helminthic parasite compounds may also be formulated to oral dosagewith conventional fillers, carriers, and excipients. The amount ofparasite administered to the individual in need thereof is an amountsufficient to prevent or treat the autoimmune disease. This amount mayvary depending upon the disease being treated or prevented and thehelminthic parasite, whether it is being administered intact, or as anegg, larvae, extract or cercariae.

Typically, when the parasites are administered for all autoimmunediseases discussed herein, the amount ranges from about 50 to about50,000. More particularly, this amount may range from about 500 to about5,000. When eggs are utilized, about 500 to about 5000 may be utilizedto treat the autoimmune diseases disclosed herein. When extracts areadministered, about 100 μg to about 10,000 μg are utilized to treat theautoimmune diseases. When larvae and cercariae are administered, thedosages may range from about 500 to about 5,000 in each case.

For prevention or vaccine use, the amounts of the parasites may be500-5,000. Any of the pharmaceutical helminthic parasite preparationsdescribed herein may be administered once or in several doses dependingon the form given, the disease and patient being treated, and theclinical evidence of a therapeutic response.

As used herein, “treatment” of a disease refers to a protocol orprocedure which reduces the presence, severity, and/or frequency of theclinical symptoms of a disease. Depending on the clinical symptoms beingmeasured and the scale used for their measurement, changes of as littleas 5% may be indicative of successful treatment. The most reliable meansof monitoring or adjusting the treatment regimen is to monitor thesymptoms of the disease being treated and the markers of inflammatoryresponses being manipulated (i.e., Th1 vs. Th2). As described above, thepresence of ova in the stool indicates successful colonization by aparasite according to the invention. An increase in cytokines indicativeof a Th2 response and/or a decrease in those cytokines indicative of aTh1 response is indicative of successful modulation of of inflammation.Any corresponding change in disease-specific symptoms will aid in theassessment of the effectiveness of the treatment approach. Methods ofevaluating Th1 and Th2 responses are described below.

D. Determination of Th1 and Th2 Responses

In order to show the efficacy of the present invention, the Th1 and Th2response must be distinguished. Metawali et al., 1996, J. of Immunol.157:4546 has shown that in mice, it is possible to distinguish a Th1from a Th2 response by histologic analysis, and by analysis of cytokineand immunoglobulin profiles. Further, Sandor et al., 1990, J. of Exp.Med. 171:2171 has shown that cell surface expression of Fcg3 and MHCClass II molecules afford discrimination. In this procedure, small boweland colon are examined histologically to determine the degree of mucosalinflammation, eosinophilia and mastocytosis. The latter cell types areindicative of a Th2 response. Mesenteric lymph nodes (MLN) and spleenscan be dissociated into single cell suspensions for in vitro culture inmicrowell plates. Cells (1-2×10⁷/well) in complete RPMI medium arecultured for up to 72 h in the presence or absence of worm antigen oranti-CD3 and then the supernatants are assayed for cytokines andimmunoglobulins. IFN-γ, TNFα and IgG2a characterize a Th1 response,whereas IL-4, IL-5, IgE and IgG1 typify a Th2 reaction. Also, serum canbe assayed for cytokine and immunoglobulin concentrations. Furthermore,dispersed inflammatory leukocytes are examined by flow cytometry forFcγ3 expression on macrophages (Th1) and MHC Class II expression on Bcells (Th2). Controls include serum, MLN and spleens from appropriateage-matched, littermate mice that hosted no parasite. Also, there areother markers of the Th1 vs Th2 responses.

A similar analysis can differentiate a human Th1 from a Th2 response.One examines inflamed tissue, isolated leukocytes from regions ofinflammation and peripheral blood cells. Leukocytes are cultured invitro alone or in the presence of parasite antigen or mitogens tostimulate cytokine release, and the cytokines are analyzed by, forexample, ELISA. The specific pattern of cytokines released allowsdifferentiation of Th1 from Th2 responses. IgG2a is generally indicativeof a Th1 response, whereas IgE, IgG1 or IgG2 are indicative of Th2response.

Cytokine Detection by Flow Cytometry: Splenocytes, MLN or intestinalinflammatory cells in RPMI complete medium are placed into 24-welltissue culture plates at 2×10⁶ cells/well. Cells are incubated 4-6 h inthe presence or absence of anti-CD3 or appropriate antigen withbrefeldin A at 10 μg/ml. Brefeldin prevents exocytosis of proteins andpromotes accumulation of the cytokine within the cell. For cytoplasmiccytokine detection, the cells are fixed in 2% paraformaldehyde at roomtemperature for 5 min following surface staining to distinguish cellsubtypes. Cells are washed and re-suspended in 50 μl PBS 0.2% Saponinand 1 μg anti-cytokine antibody and incubated at room temperature for 20minutes. Next, the cells are washed twice in Saponin and re-suspended inPBS/FCS. The specificity of the cytokine antibody staining is confirmedby pre-blocking the cells with an excess of un-conjugated antibody ofthe same isotype and cytokine specificity or by incubating the cells inthe presence of recombinant cytokine Phycoerythrin (PE)-labeledirrelevant antibody controls also are included to assess backgroundstaining. The cells are analyzed using flow cytometry.ELISAs: ELISAs measure cytokine and antibody concentrations in cellsupernatants from cells cultured in microtiter plates and manipulated asdescribe above. Cytokines are assayed using two monoclonal antibodies(mAbs) in a two-site sandwich ELISA. The anti-cytokine mAbs are purifiedby ammonium precipitation from supernatants of antibody secretinghybridoma clones. Microtiter plates are coated with 50 μl of 1 μg/mlcoating antibody in PBS containing Tween 20 (PBS-T), and incubated at 4°C. overnight. Then, wells are blocked by the addition of 150 μl of 10%FCS in PBS with incubation at 37° C. for 30 min. Standards compriserecombinant cytokine or cytokine-containing supernatants from ConA-activated spleen cells from schistosomiasis-infected mice. Sample andstandard dilutions are made in RPMI containing 10% FCS (complete RPMI)in a separate 96-well flat bottom microtiter plate, and 50 μl volumesare transferred to the ELISA plates that have been washed three times inPBS-T. Samples are incubated in the assay plates for 1 h at 37° C.Appropriate mAb is conjugated to biotin. After washing three times inPBS-T, each well will receive 50 μl of antibody-biotin conjugate at 0.5μg/ml in 1% BSA/PBS-T. Plates are incubated at room temperature for 1 hfollowed by washing three times in PBS-T. Streptavidin-horseradishperoxidase conjugate (75 μl) is added at 1 μg/ml in 1% BSA/PBS-T andincubated at room temperature for 1 h. Plates are washed 10 times infresh PBS-T, and 100 μl of substrate (2.2′-azino(3-ethylbenzthiazolinesulfonic acid) at 1 mg/ml in 44 mM Na₂HPO₄, 28 mM citric acid, and0.003% H₂O₂ is added. The colored product is measured at a wavelength of405 nm with a reference wavelength of 490 nm, using a multiscanmicroplate reader.

Immunoglobulins are quantified using anti-isotype specific ELISAs.Affinity-purified goat anti-IgM, -IgG1, -IgG2a, -IgG2b, -IgG3, -IgA and-IgE are used as capture antibodies and absorbed to flexible polyvinylmicrotiter dishes at 10 μg/ml. After addition of culture supernatants,incubation and washing, appropriate isotypealkaline-phosphatase-conjugated goat anti-Immunoglobulin is used todetect total mouse Immunoglobulin bound to the plates. Standard curvesare generated using purified Immunoglobulin. To measure parasiteantigen-specific antibody, soluble antigen is biotinylated and used todetect bound mouse Immunoglobulin. The plates are analyzed on a ELISAreader, and concentrations of total Immunoglobulin are determined usingthe standard curve and best-fit analysis software. Antigen-specificantibody concentrations are compared relative to the O.D. readings,since soluble parasite antigen is not a defined antigen that permitsprecise quantitation.

ELISPOT Assays: ELISPOT assays are established to count lymphocytessecreting either polyclonal antibody or cytokines Ninety-six-wellnitrocellulose-backed microtiter plates are coated overnight at 4° C.with 1 μg/ml of either anti-Immunoglobulin or anti-cytokine antibodiesin PBS-T. The plates then are blocked with PBS containing 10% FCS andwashed extensively with PBS-T.

Serial dilutions of a single cell suspension, starting with 5×10⁴cells/well, are incubated on the plate for 5 h at 37° C. in a humidified5% CO₂ atmosphere. The plates are washed with PBS-T and overlaid withbiotinylated anti-Immunoglobulin or -cytokine antibodies overnight at 4°C. Next, plates are washed and treated with streptavidin-glucoseoxidase-conjugate for 2 h and washed again.

The antibody or cytokine secreted by single cells is visualized withsubstrate. The colorimetric reaction is halted after 30 min by washingand spots enumerated under 30× magnification. The dilution of cellsproducing 10-50 spots/well is used to calculate the total number ofsecreting cells per sample. Controls include wells coated withinappropriate goat antibody or inappropriate antigen, or left uncoated.

A modification of the assay using soluble antigen to coat the wellspermits quantitation of parasite antigen-specific, Immunoglobulinsecreting B cells also. Briefly, for example, plates are coated withadult T. muris antigen at 0.25 μg/well or appropriate irrelevant controlantigen. Cells are added to the wells after washing. After appropriateincubation, the plates are washed again and treated as described above.

Statistical Methods: Some data are analyzed by the one sample t-test todetermine whether mean values significantly differ from zero. Pairedt-test is used to analyze differences between group means. Analysis ofvariance and Dunnett's t-test is used to analyze multiple comparisondata.Evaluation of Disease: Evidence of the presence of an autoimmune diseaseand its cure or amelioration is needed to determine the need fortreatment and to monitor treatment progress. The following proceduresare utilized to measure the clinical parameters of the noted diseases.

1. Inflammatory Bowel Disease

Evaluation of Inflammation: In mice, clinical evidence of diseaseincludes weight loss, diarrhea, rectal prolapse and histologicalevidence of intestinal inflammation. Thus, improvement in theseparameters would signify amelioration of disease.

To grade intestinal inflammation in animal models, tissue is removed,Swiss-rolled and embedded in paraffin according to standard methods. Thesections are stained with hematoxylin and eosin. The degree of colonicinflammation is graded semiquantitatively from 0 to 4 in a blindedfashion by a single pathologist using our usual standardized technique:0=no inflammation; 1=low level inflammation; 2=intermediate levelinflammation; 3=high level inflammation with wall thickening; and4=transmural infiltration, and loss of goblet cells with wallthickening.

To count mast cells, intestinal tissue samples from individual mice areprepared by the Swiss-roll technique, fixed in Carnoy's fixative,paraffin embedded and processed for staining with Alcian Blue andsafranin. Fifty adjacent fields of a given section are scanned formucosal mast cells in the lamina propria and muscle layers. Mast cellsare identified by their distinctive intracellular granular staining withAlcian Blue. All samples are evaluated blindly.

Disease activity in humans is monitored using various clinical,laboratory and histological criteria. There are several well establishedIBD disease activity indices that monitor clinical parameters likefrequency of diarrhea and abdominal pain. One particularly useful indexfor the assessment of Crohn's disease is the Crohn's Disease ActivityIndex, or CDAI (Best et al., 1976, Gastroenterology 70: 439). The CDAIincorporates 8 variables related to the disease activity and has beenused in most recent studies of therapeutic agents in Crohn's disease. Itincludes the number of liquid or very soft stools, the severity ofabdominal pain or cramping, general well-being, the presence ofextraintestinal manifestations of the disease, presence or absence of anabdominal mass, use of antidiarrheal drugs, hematocrit, and body weight.The composite score ranges from 0 to about 600. Scores below 150indicate remission and scores above 450 indicate severe illness.

A tested, accepted and disease specific quality of life questionnairealso may be administered prior to and after treatment to assesstherapeutic progress. The Irvine Inflammatory Bowel DiseaseQuestionnaire is a 32-item questionnaire. It evaluates quality of lifewith respect to bowel function (e.g. loose stools and abdominal pain),systemic symptoms (fatigue and altered sleep pattern), social function(work attendance and the need to cancel social events) and emotionalstatus (angry, depressed, or irritable). The score ranges from 32 to224, with higher scores indicating a better quality of life. Patients inremission usually score between 170 and 190.

Also, helpful are endoscopic, x-ray and histological assessment ofintestinal disease activity. C-reactive protein levels and blood cellsedimentation rate may also be monitored as systemic indicators ofinflammation.

2. Rheumatoid Arthritis

Evaluation of Inflammation:

For mice with collagen-induced arthritis, mice are examined every otherday and their paws scored as follows: 0, normal; 1, Erythema and mildswelling confined to the ankle joint or toes; 2. Erythema and mildswelling extending from the ankle to the midfoot; 3, Erythema and severeswelling extending from the ankle to the metatarsal joints; and 4,Ankylosing deformation with joint swelling. These four parameters can becorrelated with the histological changes in the arthritic joints.Treatment success results in a decrease in the arthritis score withimprovement in the histology.

For pristane-induced arthritis, joints may be measured with a micrometerto detect swelling. In humans, RA is scored by measuring joint swelling,erythema, limitation of motion and pain. Additionally, synovial fluidmay be analyzed for cytokine and inflammatory protein concentrations,and for leukocyte composition and function, according to methods knownin the art. Synovial biopsies provide tissue for histological analysisaccording to methods known in the art.

3. Lupus

Evaluation of Inflammation:

The normal development and function of the immune system criticallydepends on the removal of unwanted cells by a process called apoptosis.Cell-to-cell interactions through specific cell surface molecules andtheir receptors frequently trigger the process. One such system iscalled FAS and FAS ligand. Mice deficient in either FAS (LPR−/−) or FASligand (GLD−/−) develop an autoimmune disease like lupus.

Colonies of LPR or GLD mice are maintained in micro-isolator housingunits under specific pathogen-free conditions. These mice can developautoimmunity spontaneously, but more predictably after artificialinduction. To induce disease, 8-wk-old mice are injected with an agentlike pristane. Within two months, the mice have autoimmune disease. Manyclinical, histological and immunological criteria useful for judgingdisease induction and amelioration in both mice and humans are wellknown in the art.

4. Juvenile Insulin-Dependent Diabetes Mellitus (Type 1)

Evaluation of Inflammation:

The NOD mouse develops type 1 diabetes mellitus similar to humans due toautoimmune destruction of the pancreatic β cells. Clinical, biochemical,immunological and histological examination according to methods known inthe art allow assessment of disease induction and amelioration in mice.

5. Sarcoidosis

Evaluation of Inflammation:

In the bead embolization model of pulmonary inflammation, antigens (ie.Th1 or Th2) are coupled to Sepharose beads, which are embolized to thelungs of mice via injection into their tail veins. The animals usuallyare pre-sensitized to the coupled antigen. The immune system of the hostmounts a vigorous immune response to the offending bead. These focalinflammatory responses, which can last for several weeks, can beexamined histologically for size. Also, they can be isolated from tissueand studied for cell composition and cytokine production. Moreover,hilar lymph nodes and spleens are readily available for experimentation.

Sarcoidosis, a disease of humans, usually involves the lung.Determination of sarcoidosis and the extent of the disease may be madeaccording to methods known in the art. Pulmonary function tests canassess lung compliance and function. Also, bronchiolar lavage obtainsinflammatory cells that have infiltrated into the bronchial tree duringthe inflammatory process. These cells can be studied for composition andfunction. Pulmonary infiltrates and hilar lymphadenopathy arecharacteristic of sarcoidosis. Thus, periodic chest x-ray or CT scanscan help assess disease activity. Serologic tests, such as measurementof angiotensin converting enzyme activity according to methods known inthe art, can be used to gauge disease extent and activity.

6. Multiple Sclerosis

Evaluation of Inflammation:

Experimental autoimmune encephalomyelitis is induced in susceptible miceby repeated injection of appropriate sensitizing myelin antigens. Miceare assessed clinically according to the following criteria: 0, nodisease; 1, tail atony; 2, hind-limb weakness; 3, hind-limb paralysis;4, hind-limb paralysis and fore-limb paralysis or weakness; 5, moribund.For histological analysis, the spinal cords and brains are removed andfixed in formalin. The paraffin-embedded sections are stained andexamined under light microscopy. Dispersed splenocytes and cells fromother regions can be studied in-vitro as outlined above. Theseparameters can help measure disease amelioration or improvement.

In humans, MS disease activity is gauged by monitoring progression andremittence of neurological signs and symptoms. The most widely usedoutcomes measurement is called. The Expanded Disability Status Scale.Cerebral spinal fluid protein composition and cell content analyzedaccording to methods known in the art also may be used to monitordisease activity. Moreover, serial MRI studies show newgadolinium-enhanced brain lesions. Therefore, the clinical response oreffectiveness of treatment of MS with parasite preparations may bemeasured in a number of ways. First, the relief of MS symptoms, asgauged by improved scores on the Expanded Disability Status Scale(EDSS), indicate successful treatment. Also a decreased number of brainlesions or a lack of appearance of new lesions in serial MRI scansindicates successful treatment. Decreases or changes in the patterns ofinflammatory cells or cytokines in the CSF of MS patients undergoingtreatment are also indicative of improved inflammation status. These orother criteria may be monitored at points throughout the treatmentregimen by one skilled in the art to evaluate the success of and, ifnecessary, adjust the treatment regimen.

7. Autoimmune Thyroiditis.

Evaluation of Inflammation:

To experimentally induce disease, female PVG rats are thymectomized.They then receive four 274-rad doses of ¹³⁷Cs γ-irradiation at 2-wkintervals. Development of thyroiditis is determined histologically andby development of anti-thyroglobulin antibodies. The incidence ofthyroiditis normally is about 70% using this model.

In humans, autoimmune thyroiditis is characterized by the presence ofanti-thyroglobulin antibodies, which are readily detectable usingstandard methods known in the art. Disease status may be monitored bymeasuring the amounts of such antibodies present in the peripheralblood.

8. Colon Polyps/Colon Cancer.

Evaluation of Inflammation:

Various transgenic mice (i.e., IL-10- or Gia2-deficient mice)spontaneously develop colonic epithelial dysplasia secondary to excessTh1 stimulation within the mucosa. Development of dysplasia is assessedby histological analysis of fixed colonic tissue.

In humans, colon polyps and/or colon cancer presence, prevention orregression can be assessed via colonoscopic examination and biopsy.

9. Allergic Airway Diseases.

Evaluation of Inflammation:

A. Asthma

Asthma may be induced in mice using the model of ovalbumin-inducedhypersensitivity lung disease. In this model, mice receive injections ofovalbumin i.p. to sensitize the animals to the antigen. Afterappropriate sensitization, the mice are challenged with an aerosol ofovalbumin to induce pulmonary inflammation.

Lung inflammatory cells are obtained for analysis by mincing anddispersing lung parenchyma or by broncho-alveolar lavage. Cell cultureand intracytoplasmic flow analysis are used to analyze patterns ofcytokine expression. Cell yield and composition is indicative of thenature and severity of the inflammation. Quantitation of inflammationmay also be made by histological assessment of fixed lung tissue.

In humans, clinical examination and various pulmonary function testsknown in the art are used commonly to assess airway and lunginflammation, and thereby the severity of disease. These tests can beused to monitor the clinical response to helminthic therapy.

B. Allergic Rhinitis

Many patients have defined allergies to specific pollens, fungi oranimal dander. To monitor the disease status and/or the clinicalresponse to helminthic therapy, patients are challenged via instillationof allergens into the nose and then examined for itching, rhinorrhea andother manifestations of an allergic response.

E. Identification and Isolation of Active Helminthic Components

Helminthic parasites are complex multi-cellular organisms containingnucleic acids, peptides, proteins, lipids, glycolipids, glycoproteins,carbohydrates and their metabolites such as hormones, prostaglandins,and leukotrienes. One or more of these components may have biologicalactivities that modulate or control the immune response, including theTh1 immune response.

Immunologically active helminthic agents include the life cycle stagesof egg, larvae, cyst, and adult worm. Each of these stages may beisolated as described in the above section “Helminthic ParasitePreparations According to the Invention”. Isolated eggs, larvae,cercariae, or adult worms may be dispersed into components by grindingin a tissue homogenizer.

The homogenate is prepared in a buffer that may contain emulsifyingagents such as Triton X-100 or Tween 20/80. The buffer may containanti-oxidants such as dithioerythritol or 2-mercaptoethanol. The buffermay contain protease inhibitors such as TLCK, TPCK or other agents. Thebuffer may contain cation chelators such as EDTA or EGTA.

The homogenate may be tested for biological activity in in vivo and invitro assays. In vivo assays for the ability to modulate an immuneresponse, including the ability to reduce an excessive Th1 immuneresponse, include compounding the homogenate with a known antigen suchas purified protein derivative (PPD) from mycobacterium. PPD normallyelicits a Th1 response in mice. Active helminthic homogenate (HH), whichmay derive from worms, eggs, larvae, or cercariae, compounded with PPDor a similar antigen will cause mice to respond to that antigen with aTh2 cytokine profile as determined by ELISA for IL-4, IL-5 and IFN-γ.Prevention or diminution in cell-mediated responses such as foot padswelling may also be measured.

Other in vivo models exist that are useful for assaying helminthichomogenate or fractions of such homogenate for the ability to modulatean immune response. Helminthic homogenate can be compounded with myelinbasic protein (MBP) or PLP139-151 and mixed with CFA (adjuvant). SJL/Jmice immunized with HH/MBP will develop no or greatly attenuatedexperimental autoimmune encephalomyelitis (EAE). Also, injections of HHgiven to IL-10−/− mice may prevent or ameliorate development of colitis.Moreover, injections of HH given to Balb/c or SJL mice may induce Th2responses and prevent or ameliorate TNBS colitis. Injections of HH intoNOD mice may prevent or ameliorate development of Type 1 diabetes.Injections of HH may prevent any of the autoimmune or excessiveinflammatory diseases listed in Table 1.

In vitro models of Th2 conditioning useful for assaying the ability ofHH or fractions of HH to modulate an immune response also exist. Immunecells such as peripheral blood lymphocytes (eg. murine, monkey orhuman), splenocytes or lymph node cells, or cell lines may be exposed toHH in vitro. Cells may be analyzed for diminution of critical regulatoryfactors like IFN-γ, IL-12, or IL-18 and augmentation of IL-4, IL-5,IL-10 or TGF-β by ELISA or flow cytometry. Flow cytometry also may alsobe used to measure co-stimulatory molecule display such as B7.1, B7.2,CD40 and CD40 ligand that may be diminished by HH exposure. HH-treatedcells may also be analyzed for changes in gene expression throughanalysis of total or poly (A)⁺ RNA populations. Total RNA may beextracted from these cells and analysed for changes in transcripts forthe above cytokines, co-stimulatory molecules, cytokine receptors suchas IL-12Rβ1, IL-12Rβ2, IL-18R, IFN-γRα, IFN-γRβ, IL-2Rα, and chemokinesand their receptors. Cellular RNA may be analyzed by DNA-chipmethodology, differential display, or subtractive hybridization todiscover novel gene products regulated by exposure to HH. Isolated humanand murine cells or cell lines will be exposed to HH to determineactivity of helminthic preparations on specific cell types. Cells may bestimulated with antigens, superantigens, mitogens, phorbol esters, orsignal transduction analogs during, before or after HH exposure tomeasure the modulatory influence of HH on cytokines, chemokines,co-stimulatory molecules, receptors or specific gene products.

A helminthic homogenate (HH) may contain one or more active componentsthat regulate the immune response. HH may be fractionated by columnchromatography, HPLC, FPLC, matrix-affinity chromatography, reversephase chromatography, and/or other electrophoretic and chromatographicseparation techniques. Fractions of HH may be tested for immune activityby in vivo and in vitro assays as described above. Fractions identifiedas having immuno-modulatory activity in one or more of the assays may befurther fractionated using chromatographic separation techniques, withsubsequent assay of the sub-fractions for activity. Suchsub-fractionation may be repeated at least once, or as necessary tofurther purify the immuno-modulatory component or components of the HH.

Animal Models Useful According to the Invention

The following table (Table 1) illustrates the disease conditions thatcan be prevented or treated by the helminthic parasites in accordancewith the present invention as well as animal models established for thestudy of these diseases.

TABLE 1 TREATABLE DISEASES SOME ANIMAL MODELS 1. Inflammatory BowelDiseases TNBS colitis (Neurath et al., 1995, J. of Exp. Med. 182: 1281)IL-2 mutant mice (Ludviksson et al., 1997, J. of Immunol. 158: 104)IL-10 mutant mice (Berg et al., 1996, J. of Clin. Investigation 98:1010) TCR transgenic mice (Mombaerts et al., 1993, Cell 75: 274) CD45+ Tcells transferred into SCID mice (Powrie et al., 1994, Immunity 1: 553)2. Rheumatoid Arthritis Murine pristane-induced arthritis (Stasluk etal., 1997, Immunol. 90: 81) Murine collagen-induced arthritis (Horsfallet al., 1997, J. of Immunol. 159: 5687) 3. Insulin-dependent Diabetes(type 1) NOD mouse (Cameron et al., 1997, J. of Immunol. 159: 4686) 4.Lupus (NZWX NZB) F₁ models (Santiago et al., 1997, J. of Exp. Med. 185:65) GRD, LPR mouse (FAS mutation) (Bhandoola et al., 1994, Int. Rev. ofImmunol. 11: 231) 5. Sarcoidosis Murine Berylliosis (Pfeifer et al.,1994, Int. Archives of Allergy & Immunol. 104: 332) M. avium mouse (Chenet al., 1994, Science 265: 1237) 6. Multiple Sclerosis Experimentalallergic encephalomyelitis (EAE) in mice (Paterson, 1976, In: Textbookof Immunopathology, Second Edition, P. A. Miescher and H. J.Mueller-Eberhard, eds. (New York: Grune and Stratton), pp. 179-213) 7.Autoimmune Thyroiditis Thyroiditis in rats (Seddon & Mason, 1999, J.Exp. Med. 189: 279) 8. Colon polyps/cancer Gai2-deficient mice (Rudolphet al., 1995, Nature Genet. 10: 143); Feline colitis (Van Kruiningen etal., 1983, J. Compar. Pathol. 93: 275); Canine colitis (Van Kruiningen,1972, Gastroenterology 62: 1128) 9. Allergic Airway Diseases Mouse modelof asthma (Randolph et al., 1999, J. Immunol. 162: 2375)In addition to the use of animal models for the study of Th1-mediateddiseases and the effects of helminthic therapy, Applicants wish to notethat the use of helminths according to the invention can be expected toprevent similar non-experimental autoimmune diseases in pigs, cats,dogs, horses and cattle, and can have important ramifications foragriculture and veterinary medicine.

Mechanism of Action

This invention involves the prevention and/or treatment of autoimmunediseases in individuals by the administration of helminthic parasites,and to the prevention or vaccination and/or treatment of an excessiveimmune response or aberrant immune response in an individual due to IBD,Crohn's disease (CD), ulcerative colitis (UC), colon polyps/coloncancer, rheumatoid arthritis (RA), insulin-dependent, juvenile diabetesmellitus (DM) (type 1), lupus erythematosus (LE), sarcoidosis, multiplesclerosis (MS) autoimmune thyroiditis, allergic rhinitis and asthma byadministration of helminthic parasite to that individual.

Without being bound to any one mechanism of action, the followingmechanism is proposed.

Inflammation is a complex process involving many cell types and therelease of various immunoregulatory molecules called cytokines. Thetypes of cytokines secreted determine the nature of the inflammatoryresponse. Immune responses usually present as either Th1 and Th2.Th1-type inflammations produce large amounts of IFN-γ and TNFα, whichstimulate a strong cellular immune reaction to invading pathogens likebacteria, viruses and protozoa. A Th2-type response contains IL-4, IL-5,IgE, IgG1, mast cells and eosinophils. This type of inflammation is seencommonly in allergic reactions. Strong Th1 and Th2 responses aremutually exclusive. IFN-γ prevents expression of IL-4, and IL-4 impedesIFN-γ production, which are the two cytokines centrally important forthe Th1 and Th2 patterns of inflammation. Also there are other cytokineslike IL-10 and TGF-β that inhibit IFN-γ synthesis.

The immune response to helminthic parasites promotes Th2 responses tounrelated antigens. Infestation with helminthic parasites, which allinduce Th2-type inflammation, can modulate the Th1 immune response tounrelated concomitant parasitic, bacterial and viral infections(Kullberg et al., 1992, J. Immunol. 148:3264). Patients infected with S.mansoni mount more of a Th2-like response to tetanus toxoid immunizationthan the usual Th1 or Th0 (Sabin et al., 1996, J. Infec. Dis. 173: 269).Ethiopian immigrants with a high prevalence of helminthic infectionshave eosinophilia and a propensity to respond to PHA with Th2, ratherthan Th1 cytokines (Bentwich et al., 1996, Clin. Exp. Immunol. 103:239).

Animal experimentation supports this contention. Mice infected withMycobacterium avium develop chronic Th1-type granulomatous inflammationin the lungs and liver. Splenocytes and granuloma cells from theseinfected animals normally produce IgG2a and IFN-γ, and no IL-4 or IL-5.However, mice infected with S. mansoni after the establishment ofMycobacterium avium infection form mycobacterial granulomas containingeosinophils. Also, splenocytes and granuloma cells from co-infected micesecrete more IgG1 and much less IgG2a. The cytokines released from thesecells both constitutively or after mycobacterial antigen stimulationinclude IL-4 and IL-5, and much less than normal quantities of IFN-γ.

There are other examples. Infection of mice with S. mansoni delaysclearance of vaccinia virus and alters responsiveness to sperm whalemyoglobin. Mice also develop a Th2 response when infected with themicrofilariae, Brugia malayi, or immunized with a soluble filarialextract from this parasite. The ongoing Th2 response to this helminthantigen modulates the Th1 response to mycobacterial antigen. Moreover,Nippostrongylus brasiliensis, a murine intestinal nematode, stimulatesTh2 activity. Nippostrongylus delays kidney graft rejection in rats.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1 General Methods

Animals: Colonies of 129/SV IL-10−/− mutant mice, and appropriatecontrol animals are maintained are housed in facilities maintained as aspecific pathogen-free environment according to standard methods.

Parasite Maintenance, Animal Infection, Production of Schistosome Eggs:The maintenance of T. muris and the method used for infection are asdescribed by Else and Wakelin, 1990, Parasitology, vol. 100, part 3:479.

Schistosome eggs were harvested from the livers of schistosome-infectedhamsters and stored as described by Elliott, 1996, Methods: A Companionto Methods in Enzymology, 9:255. Five infected hamsters yield about2×10⁶ eggs.

Preparation of T. suis Eggs: The following process was used in thepreparation and harvesting of T. suis eggs. Adult T. suis worms wereisolated from the colon of pigs 7-8 wks after exposure to anexperimental inoculation of T suis eggs. Embryonated eggs were obtainedby culturing adult worms in vitro, and then the excreted eggs, separatedfrom the culture medium by centrifugation, were placed into 0.2%potassium dichromate solution at 22° C. for 5-6 wks with bubbling toobtain infective first-stage larvae. Eggs were washed twice in sterilewater by centrifugation at 1200×g for 10 min, counted, and re-suspendedin the desired amount of saline based on a calculated dose of 2,500.These eggs were stored for use in the subjects. The ova are stable forat least one year in the refrigerator. To assure infectivity, wemonitored patients for the appearance of ova in the stool aftercolonization. The number of ova in the stool is proportional to theintensity of the infestation. Also, from time to time, we infect pigswith our stored ova to assure continued infectivity.

Infection with M. avium: Mice were infected by injecting 10⁶ colonyforming units (CFU) of Mycobacterium avium (ATCC 25291)intraperitoneally. On day 60 of infection, some mice also received 35 S.mansoni cercariae to induce dual infection.

Schistosome Infection and Isolation of Ova: Some experiments used mice(18-20 g) infected for 8-9 wks with S. mansoni. Mice were infectedsubcutaneously with 40 cercariae from the Puerto Rican strain.

Granuloma Isolation and Dispersal: Granulomas form inschistosome-infected mice because of natural egg deposition, whichbegins at the 6th wk of infection. M. avium also induces granulomas. Westudy liver granulomas isolated from infected mice as described(Elliott, 1996, supra). Granulomas were dispersed to produce single-cellsuspensions. Isolated granulomas were agitated for 35 min at 37° C. in ashaker water bath in RPMI medium containing 5 mg/ml collagenase. Theresidual granulomas were sucked and expelled through a 1 ml syringe toinduce further dissociation. The resulting cell suspension was filteredthrough gauze and washed three times. Cell viability was determined byEosin-Y exclusion. This protocol resulted in a high yield of viableinflammatory cells that show preserved surface molecule expression.Spleen and MLN Dispersal: Splenocytes and MLN were dispersed by teasingand washing the tissue through a stainless steel mesh. The contaminatingRBC were lysed with H20, and the cells were washed ×3 in RPMI beforeuse.Induction of TNBS Colitis: Colitis was induced by rectal instillation oftrinitrobenzesulfonic acid (TNBS) as described by Neurath et al., 1995,J. Exp. Med., 182: 1281. Briefly, control or parasite-exposed BALB/cmice were fasted for 30 hrs then anesthetized with methoxyflurane. A 1.2mm catheter was advanced 4 cm into the colon and 0.1 ml of TNBS solution(5 mg/ml TNBS (Sigma) in 50% ethanol) was instilled. The animal was heldby the tail for 3 minutes to insure uniform contact with colonic mucosa.Evaluation of Mucosal Inflammation: To grade intestinal inflammation,tissue was removed at the indicated time points, Swiss-rolled andembedded in paraffin according to standard methods. The sections werestained with hematoxylin and eosin. The degree of colonic inflammationwas graded semiquantitatively from 0 to 4 in a blinded fashion by asingle pathologist using our usual standardize technique (19). O=noinflammation, 1=low level inflammation, 2=intermediate level, 3=highlevel inflammation with wall thickening, 4=transmural infiltration, lossof goblet cells, wall thickening.ELISAs for Murine Cytokines: ELISAs were performed as described insection VI, above.

Example 2 Th2 Response to S. mansoni Down-Modulates an Ongoing Th1Response to an Unrelated Bacterial Th1-Inducing Antigen

It is well established that Th cell immune responses can polarize intoTh1 or Th2 patterns. This polarization occurs because IFNγ from Th1cells inhibits proliferation of Th2 cells, while IL-4 and IL-10 from Th2cells inhibits development of Th I cells. The following experimentsdemonstrated that schistosomiasis alters the murine Th1 response to anestablished mycobacterial infection.

Mice were co-infected with M. avium and S. mansoni to evaluate the hostresponse to these distinctly different Th1 and Th2 inflammatory stimuli.BALB/cAnN mice develop chronic M. avium infection when injected withthis organism (10⁶ CFU). Sixty days after establishment of themycobacterial infection, the mice were infected with S. mansoni (40cercariae). The mice were killed sixty days later. Control groupsincluded mice receiving either M. avium only for 120 days or S. mansonionly for 60 days. Dispersed splenocytes or isolated granuloma cells fromthese animals were cultured in vitro (4×10⁵ cells/well) for 48 h in thepresence or absence of schistosome egg antigen (SEA, a strong Th2antigen) or mycobacterial antigens purified protein derivative (PPD, astrong Th1-inducing antigen) used at optimal concentration. After theincubation, supernatants were assayed for cytokine or immunoglobulinproduction using ELISAS. The data in FIGS. 1-3 are mean values+/−SD ofthree separate experiments.

Splenocytes from mice infected only with M. avium secreted large amountsof IFNγ following stimulation with PPD (Th1 antigen). Spleen cells fromuninfected control mice produced none. Most importantly, no IFNγ wasdetected in spleen cell cultures from concurrently infected mice (M.avium alone vs. concurrent infection, P<0.001, FIG. 1). Solubleschistosome egg antigen (SEA, Th2 antigen) stimulated only IL-4 and IL-5release from splenocytes of S. mansoni-infected animals. Mice singularlyinfected with M. avium produced no IL-4 or IL-5 in response to PPD orSEA. However, splenocytes from co-infected animals secreted some IL-4following PPD stimulation (FIG. 1).

Granulomas were isolated from the livers of mice infected with M. aviumor S. mansoni, or from animals that had concurrent infection.Concurrently infected animals develop liver granulomas that contain bothschistosome eggs and mycobacteria readily evident on histologicalexamination. Dispersed granuloma cells from these animals were culturedin vitro for 48 h in the presence or absence of SEA or PPD used atoptimal concentration. Granuloma cells from mice only infected with M.avium secreted large amounts of IFNγ following stimulation with PPD. NoIFNγ was detected in granuloma cell cultures from concurrently infectedmice (M. avium alone vs other, P<0.001) (FIG. 2). SEA stimulated IL-4release from granuloma cells of S. mansoni-infected animals. SEA did notpromote IFNγ secretion under any circumstance. Mice singularly infectedwith M. avium produced no IL-4 in response to PPD. However, granulomacells from co-infected animals secreted some IL-4 following PPDstimulation (FIG. 2).

Th1 responses promote IgG2a production, whereas Th2 reactions enhanceIgG1 and IgE. FIG. 3 shows that mice infected with M. avium have highserum IgG2a levels. Yet, co-infected animals have normal serum IgG2aconcentrations, but increased IgG1 and IgE levels. These data takentogether show that a Th2 response to a helminthic infection candown-modulate the ongoing host response to even a strong Th1-inducingorganism like M. avium.

Example 3 Colonization with Intestinal Helminths or Exposure to theirOva Attenuates Th1-Type Gut Inflammation in Murine TNBS-Induced Colitis

Rectal instillation of TNBS in 50% ethanol induces a colitis in micethat shares features with Crohn's disease. The colonic inflammation ischaracterized by infiltrating CD4+ T cells with elevated IFNγ mRNAexpression. Lamina propria T cells from TNBS treated mice secrete 50fold more IFNγ and 5 fold less IL4 than T cells from controls (Neurathet al., 1995, supra). Lamina propria mononuclear cells secrete 30 foldmore TNFα than cells from control mice (Neurath et al., 1997, Eur. J.Immunol., 27: 1743). Importantly, TNBS colitis can be prevented orimproved by treatment with anti-IL-12 (Neurath et al., 1995, supra),anti-TNFα (Neurath et al., 1997, supra), or rIL-10 (Duchmann et al.,1996, Eur. J. Immunol., 26: 934). TNBS-induced colitis also can beprevented by previous oral exposure to the hapten (Elson et al., 1996,J. Immunol., 157: 2174) probably by increasing mucosal IL-4, IL-10 andTGFβ responses (Neurath et al., 1996, J. Exp. Med., 183: 2605).

In the experiments discussed below, colitis was reproducibly induced inBALB/C mice by rectal administration of TNBS (0.1 ml of 5 mg/ml stock)in 50% ethanol. For each of the TNBS experiments discussed below,parasite-exposed and control animals were administered the samepreparation of TNBS on the same day by the same operator who was blindedto the treatment group.

A. Schistosomiasis inhibits IFNγ release from MLN and spleen cells ofTNBS treated mice.

As described above, schistosomiasis inhibits the murine Th1 response toan established mycobacterial infection. We extended these observationsto determine if schistosome infection alters the Th1 response in TNBStreated mice. Mice were infected with 35 S. mansoni cercariae by sc.injection. Worms mature and begin to lay eggs about 6 weeks afterinitiation of infection. Two weeks later (at 8 wks of infection), micewere treated with TNBS. The capacity of MLN and spleen cells to makeIFNγ in response to T cell stimulation (anti-CD3) was examined severaldays later. As shown in Table 2, natural schistosome infection stronglyinhibits IFNγ release from mesenteric lymph node (MLN) and spleen cellsof TNBS-treated mice.

TABLE 2 Schistosomiasis Inhibits IFNγ Production from MLN and Splenic TCells of TNBS-Treated Mice α-CD3 stimulated IFNγ (ng/ml) Group MLN cellsSpleen cells Uninfected mice 3.2 ± 0.7 44.1 ± 2.3 given TNBSSchistosome-infected 1.9 ± 0.3^(a)  4.2 ± 0.4^(b) mice given TNBS MeanIFNγ (ng/ml) ± SE of triplicate determinations measured by ELISA. ^(a)p< 0.1 ^(b)p < 0.05 Cultures contained 10⁶ dispersed MLN or Spleencells/well incubated in 200 μl medium for 48 hrs at 37° C. in thepresence of anti-CD3 (1 μg/ml) (2C11). Results are representative of twoexperiments.B. Exposure to schistosome ova inhibits IFNγ release from MLN and spleencells of TNBS treated mice.

In schistosomiasis, it is exposure to the parasite ova rather than adultworms that induces a Th2 response. Schistosome infection does not inducestrong Th2 responses until the worms mature and begin to lay eggs(Grzych et al., 1991, J. Immunol., 146: 1322). Mice exposed to intactschistosome eggs in the absence of natural infection develop a strongTh2 response (Oswald et al., 1994, J. Immunol., 153: 1707). Theseobservations suggest that exposure to schistosome eggs in the absence ofnatural infection may induce Th2 and suppress Th1 responses.

The hypothesis that pre-exposure to schistosome eggs would inhibit Th1responses without requiring infection by adult worms was tested. Micewere inoculated twice with 10⁴ schistosome eggs by intraperitoneal (ip)injection at 14 and 4 days prior to rectal challenge with TNBS. Thesetimes were chosen to model the continuous egg deposition that occurs innatural infection. Mice not exposed to parasite eggs but treated withTNBS served as controls. The eggs were previously frozen and were notviable at the time of injection. Once again, the capacity of MLN andspleen cells to make IFNγ in response to T cell stimulation (anti-CD3)was examined several days after TNBS instillation. Like naturalschistosome infection (Table 2), ip. egg exposure inhibited IFNγproduction from MLN and splenic T cells of TNBS-treated mice as shown inTable 3.

TABLE 3 Exposure to Schistosome Eggs Inhibits IFNγ Production from MLNand Splenic T Cells of TNBS-Treated Mice α-CD3 stimulated IFNγ (ng/ml)Treatment MLN cells Spleen cells TNBS alone 4.65 ± 0.02 47.1 ± 3.0 Eggsip. and TNBS 2.19 ± 0.11^(a) 21.8 ± 3.4^(b) Mean IFNγ (ng/ml) ± SD oftriplicate determinations measured by ELISA. ^(a)p < 0.5. Culturescontained 10⁶ dispersed MLN or spleen cells/well incubated in 200 μlmedium for 48 hrs at 37° C. in the presence of anti-CD3 (1 μg/ml)(2C11). Results are representative of three separate experiments.C. Exposure to schistosome ova protects mice from TNBS-induced colitis.

Inhibiting development of the mucosal Th1 response can attenuateTNBS-induced colitis. Prior exposure to schistosome eggs inhibits Th1cytokine secretion by MLN and splenic T cells. It was next ascertainedif ip injection of schistosome eggs would inhibit TNBS-induced colitis.Eggs were injected as above followed by TNBS treatment. Egg treatmentdramatically reduced cumulative mortality in three separate experimentsfrom 60% (16/27) in the control group to 22% (6/27) in egg-exposed mice.Intestinal inflammation was scored on a 4-point scale as detailed inGeneral Methods, above. In the mice that survived, egg treatmentattenuated intestinal inflammation from 3.1±0.5 (mean±SD) in the controlgroup to 1.3±0.3 in the egg exposed mice (p<0.05, FIG. 4). Subsequentexperiments showed that the largest difference between groups wasevident at 3 days after TNBS treatment. Other experiments carried out to14 days after TNBS instillation showed that egg exposure affordedprolonged protection. These data indicate that schistosome eggs protectmice from developing fatal colitis by inhibiting mucosal Th1 responses.

D. Intestinal elminths induce host Th2 responses.

It is probable that helminthic parasites other than S. mansoni canmodulate host Th1 responses. Expression of protective immunity tointestinal nematodes is CD4 T cell-dependent. Mice expel worms or limitinfection by mounting a Th2 response. Worm expulsion does not appear toexclusively depend on intestinal eosinophilia nor mucosal mastocytosis.IL-4 may have a critical role in worm expulsion, since treatment withblocking anti-IL-4 or anti-IL-4 receptor mAb promotes worm retention(Else et al., 1994, J. Exp. Med., 179: 347). Conversely, treatment withIL-4 promotes worm clearance (FIG. 4).

T. muris lives in the colon of the murine host. It is related toTrichuris trichiura, a parasite carried by nearly one billion peopleduring their lives (Grencis et al., 1996, Gastroenterology Clinics ofNorth America, 25: 579). Ingestion of eggs initiates infection. The eggsrelease larvae that penetrate the cecal epithelium. They then matureinto adult worms. The parasite does not replicate within the host,allowing control of the intensity of infection (Bancroft et al., 1994,Eur. J. Immunol., 24: 3113).

T. muris was used to down-modulate intestinal Th1 responsiveness. T.muris infection is established by oral gavage with 250 embryonated eggscontaining viable larvae. BALB/c mice can harbor T. muris and naturallyexpell the worms within 4 wks of infection. T. muris- or sham-infectedmice were treated with rectal instillation of TNBS 4 wks afterinitiation of infection. Prior colonization with T. muris reducedcumulative mortality in two separate experiments from 58% (7/12) in thesham infected group to 21% (3/14) in the parasite exposed group.Furthermore, mice previously colonized with T. muris develop attenuatedTNBS colitis (0.92±0.5, mean±SD) as compared to sham-infected mice(3.13±0.63, p<0.05). These data indicate that prior exposure tointestinal parasites (T. muris) protect mice from developing severeTh1-mediated colitis.

Example 4 IL-10 gene disruption does not significantly alter thehost/parasite interaction

IL-10 is an important immunoregulatory cytokine that down modulatesmacrophage activation and accessory cell function (Moore et al., 1993,Ann. Rev. Immunol., 11: 165). Mice rendered IL-10 deficient by targetedgene disruption (IL-10−/−) develop a chronic enterocolitis that isinfluenced by colonic flora (Kuhn et al., 1993, Cell, 75: 263). Theintestinal inflammation is attenuated by treatment with anti-IFNγantibody demonstrating that the colitis results from overly exuberantTh1 responses to colonic contents (Berg, et al., 1996, J. Clin. Invest.,98: 1010). These mice serve as excellent models for spontaneous colitissimilar to that of Crohn's disease. In this example IL-10−/− mice on the129 and C57B1/6 backgrounds were used.

A. Distruption of the IL-10 gene does not alter host susceptibility toparasite colonization.

IL-10−/− mice spontaneously develop chronic intestinal inflammation. Apotential concern was that this inflammation might prevent effectivecolonization with T. muris. It was found, however, that patentcolonization occurred routinely (>95%) with T. muris in IL-10−/− mice onboth the 129 and C57B1/6 backgrounds. To date, more than 40 IL-10−/−mice have been colonized with T. muris. IL-10 deficient mice also canharbor S. mansoni that mature to adult worms and lay eggs (Wynn et al.,1998, J. Immunol., 160: 4473). These findings have been confirmed (seebelow).

B. IL-10−/− mice can mount Th2 responses to parasites.

Mice infected with the intestinal nematode N. brasiliensis developTh2-type inflammation to the parasite with production of IL-4, IL-5 andIL-10. N. brasiliensis can colonize IL-10−/− mice and stimulate anappropriate intestinal Th2 response (Kuhn et al., 1993, supra). IL-10−/−mice were infected with S. mansoni to examine whether they mount a TH2response.

Table 4 shows that splenocytes from IL-10 gene-disrupted mice colonizedfor 8 wk with S. mansoni secrete large amounts of IL-4 upon stimulationwith schistosome egg antigen (SEA) or anti-CD3. The granulomas thatsurround schistosome eggs in these mice contain the usual highpercentage (45-50%) of eosinophils, and make IL-4 and IL-5. Thus, theyshow an effective Th2 response. These data indicate that exposure tohelminthic parasites like S. mansoni will induce a strong Th2 responseeven in the absence of IL-10.

TABLE 4 Schistosomiasis Induces a Th2 Response in IL10 Deficient MiceSpleen Cells from Schistosome-infected Mice SEA- α-CD3 CytokineUnstimulated Stimulated Stimulated IL-4 (ng/ml) Wild Type 0.07 ± 0.080.21 ± 0.02 3.94 ± 0.12 IL-10−/− 0.41 ± 0.10 2.56 ± 0.23 5.06 ± 0.33Mean IL-4 (ng/ml) ± SD of triplicate determinations as measured byELISA. Cultures contained 10⁶ cells/well incubated for 48 hrs at 37° C.in 200 μl medium in the absence or presence of schistosome egg antigen(SEA 5 μg/ml) or anti-CD3 (1 μg/ml) (2C11). Results are representativeof two separate experiments using a minimum of four wild type and KOmice in each experiment.

C. Helminthic Th2-conditioning inhibits natural development of mucosalinflammation in IL-10−/− mice.

It was shown that IL-10 deficient mice could harbor helminthic parasitesand mount a strong Th2 response. Because IL-10−/− mice can develop Th2responses and harbor intestinal parasites, they serve as excellentmodels to study the effect of parasite exposure on spontaneous orongoing colitis. IL-10 is an important anti-inflammatory cytokine. It ispossible that disruption of this essential immunoregulatory circuit willprevent mucosal Th2 conditioning by parasites. Present evidenceindicates that infection with T. muris impedes the spontaneous colitisthat develops in IL-10−/− mice. Animals (6-wk-old) received T. muris orsham infection and were killed 6 wks later. Colonic inflammation of T.muris or sham-infected IL-10−/− mice was scored on a 4-point scale asdetailed in General Methods, above. Prior infection with T. murisattenuated intestinal inflammation from 3.0±0.3 (mean±SE) in thesham-infected group to 2.2±0.1 (p<0.05) in the parasite exposed IL-10−/−mice. These data demonstrate that prior exposure to helminthic parasitesattenuates spontaneous colitis in IL-10 deficient mice.

Example 6 Intestinal Colonization with Whipworm Down-Modulates DiseaseActivity in Patients with Crohn's Disease

The major hypothesis of the present invention is that failure to obtainhelminthic infections and experience mucosal Th2 conditioning duringchildhood predisposes to Crohn's disease and other autoimmune diseaseswhich are the result of overly active Th1 reactions. An importantcorollary hypothesis is that colonization with intestinal parasites willdown-modulate established inflammation. It is expected that this ispossible because concurrent schistosomiasis impedes Th1 responses in anestablished M. avium infection (see above). This hypothesis was testedby colonizing patients with Crohn's disease with Trichuris suis andassessing improvement in disease activity. T. suis, the porcinewhipworm, is closely related to T. trichiura, a human intestinalhelminth common in underdeveloped countries. The whipworm is a potentialagent for therapy. Any mammalian whipworm is contemplated for useaccording to the invention. The natural human parasite Trichuristrichiura is a very small organism that resides in the colon byattachment to the mucosa. Ordinary colonization usually produces nosymptoms and causes no health problems for the host. This is the case inmillions of colonized people throughout the world, but in a minority,heavy infestation produces diarrhea, bleeding and iron deficiencyanemia. It is of interest that the parasite's life cycle is such thatthe host is not self-infected. That is, the eggs require a soil phasefor maturation to become infective and then must be re-ingested toincrease the parasitic load for an individual. Thus, T. trichiurainfestation will not increase within the host unless eggs in the soilare ingested. The agent is readily and effectively treated via a threeday course of treatment using mebendazole.

The porcine whipworm is closely related to the species infecting humans.The two organisms are of the same family and are morphologicallysimilar, but they belong to a different species and can be distinguishedmorphologically, developmentally and clinically. The porcine whipwormovum is slightly larger, has a different shaped spine, and the rate ofdevelopment from an egg to an adult is slower than that of Trichuristrichiura (Beer, 1976, Res. Vet. Sci., 20:47). Importantly, infectiveparasite ova can be obtained from SPF animals. Because the humanwhipworm is grown in humans and thus may carry with it the potential totransmit other infectious agents from the host, the procine whipworm wasused here.

A supply of infective T. suis eggs was produced as described above. Theeggs were tested to confirm absence of contamination with entericpathogens (e.g. Shigella, Salmonella, Campylobacter, Yersinia, andenterotoxic E. coli) and viruses (e.g. CMV, HSV, VZV, adenovirus andenteroviruses).

Four patients with advanced, medication-resistant (i.e., refractory)Crohn's disease were colonized with T. suis. They tolerated the parasitewith little or no symptoms attributable to this organism. Table 5 showsthat after achieving patent colonization, all participants had improvedscores on each of the different disease score indices within 2-3 weeks.That is, a drop in Crohn's Disease Activity Index (CDAI; a drop below150 on this index is considered a complete remission), and inflammatoryindices (i.e., erythrocyte sedimentation rate (ESR) and C-reactiveProtein level) was detected in all cases. Similarly, an increase in theinflammatory bowel disease quality of life indicator (IBDQ), in whichhigher scores indicate improved quality of life, was seen in all cases.These results indicate that helminths are useful for modulating abnormalimmune responses, including, but not limited to Crohn's disease.

TABLE 5 Patients with Crohn's Disease Benefit From Colonization with T.suis. C-reactive CDAI IBDQ ESR Protein Patient Pre Post Pre Post PrePost Pre Post 1 250 147 115 198 19 6 0.9 <0.5 2 341 19 127 221 6 1 <0.5<0.5 3 176 135 118 175 41 34 9.1 2.4 4 565 413 54 104 30 20 0.7 <0.5

Example 7 Intestinal Colonization with Whipworm Down-Modulates DiseaseActivity in Patients with Ulcerative Colitis

In addition to Crohn's disease, the present invention is useful for thetreatment of ulcerative colitis. Two patients with ulcerative colitiswere treated with a single dose of the organism T. suis in the samemanner as those treated for Crohn's disease above. As shown in Table 6,when evaluated at times similar to those in treatment of Crohn'sdisease, both patients experienced increases in IBDQ, and reductions inthe number of stools per week, ESR and C-reactive Protein levels. Thesedata indicate that helminths are of use for the treatment of ulcerativecolitis.

TABLE 6 Patients with Ulcerative Colitis Benefit from Colonization withT. suis. Stools/ C-reactive IBDQ Week ESR Protein Patient Pre Post PrePost Pre Post Pre Post 1 138 191 42 26 14 9 0.9 <0.5 2 166 188 16 9 1919 0.9 0.6

Example 8 Helminths Protect in a Murine Model of Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating diseaseof the central nervous system (CNS) involving autoimmune CD4+ helper Tcells, particularly cells of the T helper 1 (Th1) subgroup. It has beenrecognized for some time that there is a striking inverse correlation inthe occurrence of parasitic and autoimmune diseases. The goal of thisproject was to test whether this inverse correlation is coincidental orcausative. MS is most prevalent in temperate regions that are highlyindustrialized. This suggests that there is some critical environmentalfactor responsible for the change in frequency. It is herein proposedthat the absence of exposure to intestinal helminthic infections inchildhood is an important environmental factor.

Several animal models have been developed to study MS in animals. Thesemodels include cuprizone-induced demyelination, infection with virus andExperimental Allergic Encephalomyelitis (EAE). Of these various models,EAE has been the most studied and has served for many years as anexcellent animal model for studying the pathogenesis of MS.

EAE is an experimental autoimmune inflammatory demyelinating disease ofthe CNS and serves as an animal model to study demyelinating diseases ofthe CNS. It has three forms, which vary in clinical course andneuropathology: acute EAE, hyperacute EAE and chronic relapsing EAE.Acute EAE and hyperacute EAE are monophasic diseases, which resemble thehuman diseases acute disseminated encephalomyelitis and acutehemorrhagic leukoencephalitis, respectively. Chronic relapsing EAE has arelapsing course and is most similar to MS. EAE is induced bysubcutaneous injection of central nervous tissue homogenate or antigensemulsified in complete Freund's adjuvant (CFA) into susceptible animals.Several antigens have been shown to be encephalitogenic, such as myelinbasic protein (MBP) and myelin proteolipid protein (PLP).

Studies Using the EAE Model of Multiple Sclerosis

1. Schistosome ova injection significantly ameliorates the clinicalcourse of PLP-induced EAE in SJL/J mice.

In the first experiments, two experimental protocols have been optimizedto characterize the effect of schistosome ova injection on EAE. The twoprotocols are different only in the frequency of high or low doseimmunization with schistosome ova. In Protocol #1, 6-8 week old femaleSJL/J mice were injected intraperitoneally with 10,000 schistosome ovafourteen days prior to EAE induction. Schistosome ova injection wasrepeated at day 4 prior to EAE induction using 5000 schistosome ovaintraperitoneally and 5000 subcutaneously. This protocol had been shownto induce a very strong Th2 type immune response. On the day of EAEinduction (day 0), the experimental animals were injected bysubcutaneous tail base injection of 50 ug of PLP139-151 (HSLGKWGHPDKF)peptide in CFA containing 1 mg of heat inactivated mycobacteriumtuberculosis (H37RA; DIFCO Labs, Detroit, Mich.). 200 ng of Pertussistoxin (LIST Biological Labs, Inc., Campbell, Calif.) was also injectedon days 0 and 2.

In Protocol #2, two additional subcutaneous injections of 5000schistosome ova were included on days 0 and 7.

In both protocols, PLP139-151 peptide immunization was repeated a secondtime, 7 days following the primary injection. Animals were subsequentlymonitored for the development of clinical signs of paralysis

The results showed that both protocols were effective in inhibiting theclinical course of EAE (disease severity was scored using the scaledescribed herein under “Multiple Sclerosis: Evaluation ofInflammation”), however, the more frequent injection with schistosomeova resulted in a much stronger response, with almost completeresistance to EAE. Animals treated according to Protocol #1 demonstrateda significantly inhibited disease peak severity compared to experimentalanimals without schistosome ova injection (FIG. 5). Disease course wasfollowed only up to day 24. The onset of disease that occurred on day 13showed no delay in EAE induction due to schistosome ova injection. Threeindependent experiments were performed.

When the frequency and dosage of schistosome eggs injected into theexperimental animals was increased (Protocol #2), a significantlyinhibited clinical score of EAE was observed (FIG. 6).

To further study the relapsing phase in the course of EAE in theseexperimental animals, the clinical score of disease was monitored out to42 days in subsequent experiments. There was no relapsing EAE responsein schistosome ova-injected experimental animals (FIG. 7). Based onthese experiments, we are convinced that parasitic infections can reducethe clinical score in experimental allergic encephalomyelitis.

2. Helminths protect the CNS from inflammatory cell infiltration.

It was asked whether the ameliorated clinical score in EAE is reflectedin a decreased number of cells infiltrating the CNS of schistosomeova-treated EAE animals.

Immunohistochemistry analysis was performed to examine inflammatory cellrecruitment to the CNS. No inflammatory cells were detected in the CNSof mice when EAE is induced with PLP in SJL/J mice concurrent withschistosome ova injection.

Specifically, to characterize whether the ameliorated clinical score inEAE is reflected in a decreased number of cellular infiltrates inschistosome ova treated EAE animals, EAE was induced with or withoutschistosome ova immunization according to Protocol #2. As expected, thisprotocol resulted in decreasing clinical scores of EAE. To study theformation of CNS inflammatory cells, tissues from the central nervoussystem were taken at day 14 after immunization. Histopathologicalanalysis of spinal cord from normal or schistosome-injected mice withEAE after immunization with PLP139-151 peptide showed significantlyameliorated cellular infiltration in animals with EAE followingschistosome ova immunization (FIG. 8). Similar results were obtained bycharacterization of brain tissues. These results further demonstratethat schistosome ova can influence Th1 type inflammatory reactions inthe CNS.

3. Helminths Protect Through Induction of Th2 Cytokines.

Additional studies were performed to characterize the mechanism ofschistosome ova-induced inhibition of clinical score and cellularinfiltration in animals with EAE. Following immunization withschistosome ova, there was up-regulation of IL-4 (FIG. 9A) anddown-regulation of IL-2 (FIG. 9B) and IFN-γ cytokine production (FIG.9C) from cells of the spleen in mice with PLP-induced EAE.

In order to investigate the mechanism of schistosome ova-inducedprotection from EAE, studies focused on evaluating the delicate balancebetween the different types of helper T cells throughout the course ofEAE were performed. It is well known that the immune system, whenchallenged with antigen, mounts either a Th1- or a Th2-type inflammatoryresponse. Th1 is associated with a vigorous cellular immune reactionproducing large amounts of IFN-γ or TNF-α. This pattern of response mosteffectively controls viral and bacterial pathogens. Th2-typeinflammation often generates IL-4 and IL-5. It already has beenestablished that colonization with helminthic parasites will induceTh2-type inflammation. Based on this information, experiments weredesigned to ascertain if the parasitic infection induces an appropriateTh2 host response that resolves upon worm expulsion.

In initial experiments, spleen cells were dissociated into single cellsuspensions for in vitro culture in microwell plates. Cells(1-2×10⁷/well) in complete RPMI medium were cultured for up to 72 h inthe presence or absence of schistosome soluble egg antigen (SEA) oranti-CD3. The supernatants were assayed for IFN-γ, IL-2 and IL-4production to ascertain if the resulting sensitization to the parasiteantigens favored Th2 development. Controls included spleens fromappropriate age-matched, littermate mice that hosted no parasite butwere induced for EAE. IL-2, IFN-γ and IL-4 cytokine production wereexamined on days 0, 13 and 45 of EAE induction. Two experimental groupswere established for this study. Experimental group (1) included femaleSJL/J mice (6-8 weeks old) that hosted no parasite but were induced forEAE. Experimental group (2) included animals immunized with schistosomeova at 14 and 4 days prior to EAE induction, according to Protocol #2,described above. Throughout the course of EAE, spleen cells fromschistosome ova-injected animals produced high levels of IL-4 and verylow levels of IL-2 and IFN-γ cytokines (FIGS. 9 and 10). Control cellsfrom EAE mice that were not exposed to ova made large amounts of IFN-γand IL-2, but little IL-4. These results suggest a crucial role for Th2cells in the development of parasite induced-protection.

Other Embodiments

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims.

We claim:
 1. A method of treating multiple sclerosis in an individual inneed of treatment comprising administering to said individual apreparation of Trichuris suis eggs in an amount sufficient to treatmultiple sclerosis in said individual and monitoring said individual fora change in at least one symptom associated with multiple sclerosis. 2.The method of claim 1, wherein said Trichuris suis eggs are raised in aspecific pathogen-free environment.
 3. The method of claim 1, whereinsaid symptom is a neurological symptom.
 4. The method of claim 3,wherein said neurological symptom is paralysis.
 5. The method of claim1, wherein said symptom is cerebral spinal fluid protein composition. 6.The method of claim 5, wherein said cerebral spinal fluid proteincomposition includes at least one cytokine.
 7. The method of claim 6,wherein said cytokine is selected from the group consisting of IL-4,IL-2, and IFN-γ.